RNA buffers the phase separation behavior of prion-like RNA binding proteins

Abstract

Prion-like RNA binding proteins (RBPs) such as TDP43 and FUS are largely soluble in the nucleus but form solid pathological aggregates when mislocalized to the cytoplasm. What keeps these proteins soluble in the nucleus and promotes aggregation in the cytoplasm is still unknown. We report here that RNA critically regulates the phase behavior of prion-like RBPs. Low RNA/protein ratios promote phase separation into liquid droplets, whereas high ratios prevent droplet formation in vitro. Reduction of nuclear RNA levels or genetic ablation of RNA binding causes excessive phase separation and the formation of cytotoxic solid-like assemblies in cells. We propose that the nucleus is a buffered system in which high RNA concentrations keep RBPs soluble. Changes in RNA levels or RNA binding abilities of RBPs cause aberrant phase transitions.

Liquid‐liquid phase separation and fibrillation of the prion protein modulated by a high‐affinity DNA aptamer

“…Specifically, at 5:1 rPrP 90-231 :aptamer stoichiometry, the number of droplets/0.1 mm 2 was 98.33 ± 9.46 for A1 and 73.66 ± 14.97 for A2, respectively, versus 12.33 ± 5.31 for rPrP 90-231 in buffer without aptamers ( Figure 8D). 21 Therefore, our data agree with data for other NA-binding proteins that undergo LLPS. It was previously shown that LLPS is highly dependent on the protein:NA molar ratio, where lower ratios of specific NAs promote phase separation.…”

“…21,44 Relevantly, A1 and A2 kept rPrP 90-231 condensates in a dynamic state, preventing transition to solid-like structures in the timeframe examined. 21,44 Relevantly, A1 and A2 kept rPrP 90-231 condensates in a dynamic state, preventing transition to solid-like structures in the timeframe examined.…”

“…8,16,17,19 Other aptameric sequences that were able to bind β-sheet-enriched PrP Sc structures can be used in the diagnosis of TSEs. Considering that reversible liquid-liquid phase separation (LLPS) is a hallmark of RNA-binding proteins, including proteins related to degenerative disorders, 15,20,21 and that it is highly dependent on the protein:NA molar ratio, 21 we investigated whether A1 and A2 could induce rPrP 90-231 LLPS. Here, we used SELEX to select two 25-mer single-stranded DNA aptamers (herein referred as A1 and A2) against the globular domain of murine recombinant PrP (rPrP 90-231 ).…”

“…21 Our main goal was to shed light on the role of NAs in prion protein misfolding and to describe PrP:NA interaction leading to LLPS and modulation of this effect depending on the NA structure and binding stoichiometry. 21 Our main goal was to shed light on the role of NAs in prion protein misfolding and to describe PrP:NA interaction leading to LLPS and modulation of this effect depending on the NA structure and binding stoichiometry.…”

Photoswitchable Phase Separation and Oligonucleotide Trafficking in DNA Coacervate Microdroplets

“…

Coacervate microdroplets produced by liquid-liquid phase separation have been used as synthetic protocells that mimic the dynamical organization of membrane-free organelles in living systems.A chieving spatiotemporal control over droplet condensation and disassembly remains challenging. [3,4] Microdroplets produced in vitro by associative (coacervates) or segregative (aqueous two-phase systems) liquid-liquid phase separation have recently been used as synthetic models of dynamic protocells. The droplets disassemble and reassemble under UV and blue light, respectively,d ue to azobenzene trans/cis photoisomerisation.

…”